Method for Detecting and Quantifying Rare Mutations/Polymorphisms

ABSTRACT

The present invention is directed to a method for detecting and quantifying rare mutations in a nucleic acid sample. The nucleic acid molecules under investigation can be either DNA or RNA. The rare mutation can be any type of functional or non-functional nucleic acid change or mutation, such as deletion, insertion, translocation, inversion, one or more base substitution or polymorphism. Therefore, the methods of the present invention are useful in detection of rare mutations in, for example, diagnostic, prognostic and follow-up applications, when the targets are rare known nucleic acid variants mixed in with the wildtype or the more common nucleic acid variant(s).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Ser No. 60/545,382 filed Feb. 18, 2004, thecontent of which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to methods of detecting and quantifyingrare nucleic acids changes, mutations or polymorphisms in a nucleic acidsample; namely, the sample contains a much smaller percentage of thechanged, mutated or polymorphic nucleic acid molecule compared to thatof the wildtype or more common variants or control nucleic acidmolecules.

Detection of a nucleic acid containing a rare polymorphism or mutationcan be problematic. Such problems occur in numerous situations, forexample, if a nucleic acid sample is suspected of containing a smallpopulation of mutant nucleic acids such as in diagnosis or prognosis ofcancer, viral infections, variations in viral infections, such asvarious HIV strains in the same individual, and the like. In all thesecases, it is important to know accurately whether the nucleic acidsample actually contains the rare mutant allele or not, and in manycases it would be helpful to know how much mutant allele containingnucleic acid is present in the sample, particularly in relation to thewildtype or the more common nucleic acid molecule.

Methods for detection and quantification of nucleic acids that containdifferences which are present in only low quantities or smallpercentages compared to a wildtype or control nucleic acid molecule inthe sample can be important in many clinical applications. Non-limitingexamples of applications wherein detection of rare nucleic acid changeswould be useful include early benign or malignant tumor detection,prenatal diagnostics particularly when using a plasma or serum DNAsample from the mother, early viral or bacterial disease detection,environmental monitoring, monitoring of effects of pharmaceuticalinterventions such as early detection of multi drug resistance mutationsin cancer treatment. Also, a number of mutations causing inheriteddiseases result in reduction of the transcript levels. Therefore,improved methods allowing detection of the mutant transcript which ispresent at very low levels would allow a simplification of mutationdetection, particularly at the RNA level, in cases wherein the mutanttranscript levels are low.

Detection of rare mutations could also provide tools for forensicnucleic acid sample analysis by providing a system to reliably detectpresence or absence of specific nucleic acid polymorphisms to provideevidence to exonerate a crime suspect.

Additionally, detection of rare mutations in biological agents such asbacteria and viruses that can be used as a biological warfare agentswould provide an important tool for detecting spread of harmfulbiological materials.

Another problem requiring a satisfactory solution is in the commonlyused genotyping methods, is a so called “allele dropout”-problem whichhappens when one allele is poorly amplified or detected and aheterozygotic allele is mis-called as a homozygote. The dropout alleleis usually, but not always, the allele that produces a higher molecularweight base extension product. A method which would allow the detectionof allele dropout, particularly in clinical diagnostic applications,would be extremely useful and improve the accuracy of distinguishingheterozygotes from homozygotes, which can be crucial for evaluating, forexample, disease prognosis.

The methods for mutation detection and nucleic acid moleculequantification have traditionally included Southern-blot andNorthern-blot hybridization, ribonuclease protection assay, andpolymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR)based methods. However, both direct detection methods and PCR-basedmethods to detect nucleic acid molecules suffer from lack of sensitivityto detect or amplify the rare nucleic acid mutation, when the samplenucleic acid contains both a large amount of the wildtype nucleic acidsand a much smaller amount of the rare mutation or polymorphism.

Absolute quantification of nucleic acid molecule copy numbers in asample is a requirement if one wishes to monitor the number of mutant orpolymorphic nucleic acids, for example, at different time points or as aresponse to a pharmaceutical intervention. However, quantification ofnucleic acid copy numbers for rare mutations is difficult using PCRbased methods because the common nucleic acid molecule is also amplifiedexponentially and the mixture of amplified sample almost always containslarge amounts of the wildtype or “normal” nucleic acid variant relativeto the rare nucleic acid variant.

A number of quantitative PCR based methods have been described includingRNA quantification using PCR and complementary DNA (cDNA) arrays (Shalonet al., Genome Research 6(7):639-45, 1996; Bernard et al., Nucleic AcidsResearch 24(8):1435-42, 1996), solid-phase mini-sequencing technique,which is based upon a primer extension reaction (U.S. Pat. No.6,013,431, Suomalainen et al. Mol. Biotechnol. June; 15(2):123-31,2000), ion-pair high-performance liquid chromatography (Doris et al. J.Chromatogr. A May 8; 806(1):47-60, 1998), 5′ nuclease assay or real-timeRT-PCR (Holland et al. Proc Natl Acad Sci USA 88: 7276-7280, 1991), andreal competitive RT-PCR (Ding et al. Proc Natl Acad Sci USA100:3059-3064, 2003).

Methods using PCR and internal standards differing by length orrestriction endonuclease site from the desired target sequence allowingcomparison of the standard with the target using gel electrophoreticseparation methods followed by densitometric quantification of thetarget have also been developed (see, e.g., U.S. Pat. Nos. 5,876,978;5,643,765; and 5,639,606). These methods, also sometimes referred to asStaRT-PCT, have severe limitations in measuring an absolute transcriptquantity in a biological sample. Because of the size differences betweenthe standard and the target sequence, the PCR amplification can not beexpected to be the same for both the standard and the target sequence.Further, because a separate gel electrophoretic separation and/orrestriction endonuclease digestion followed by gel electrophoreticseparation, and densitometric measurement are required afteramplification, the method has steps which are prone to errors and makethe quantification of small amounts of nucleic acids cumbersome.

Therefore, it would be useful to develop a method which allows sensitiveand accurate detection and quantification of nucleic acids containingrare changes and which can be easily automated and scaled up toaccommodate testing of large numbers of sample and which overcome thesensitivity problems of nucleic acid detection. Such a method wouldenable diagnosing different pathological conditions, including viruses,bacteria and parasites, as well as different benign and malignanttumors, neurological disorders, heart disease and autoimmune disorders.Such a method would also allow quantifying the rare transcripts ofinterest for diagnostic, prognostic and therapeutic purposes.

SUMMARY OF THE INVENTION

The present invention is directed to a method for detecting andquantifying rare mutations in a nucleic acid sample. The nucleic acidmolecules under investigation can be either DNA or RNA. The raremutation can be any type of functional or non-functional nucleic acidchange or mutation, such as deletion, insertion, translocation,inversion, polymorphism and one or more base substitution. Therefore,the methods of the present invention are useful in detection of raremutations in, for example, diagnostic, prognostic and follow-upapplications, when the targets are rare known nucleic acid variantsmixed in with the wildtype or the more common nucleic acid variant(s).

In one embodiment, the invention provides a method of detecting nucleicacids with a rare mutation comprising the steps of amplifying a nucleicacid molecule with at least two primers flanking the mutation site,designing a detection primer so that the 3′ end of the detection primeris immediately adjacent to the first nucleic acid which differentiatesthe wildtype nucleic acid variant from the mutant nucleic acid variantmolecule, removing the excess dNTPs after the amplification reaction,performing a primer extension reaction using the detection primer and atleast one dNTP or ddNTP, which corresponds to a nucleoside adjacent tothe detection primer in the rare mutant nucleic acid molecule, whereinthe presence of a primer extension product in the reaction indicates thepresence of the nucleic acid with a rare mutation. In the preferredembodiment, only one dNTP or ddNTP is used. However, so long as theprimer is designed so that the background wildtype or the more commonnucleic acid molecule(s) cannot serve as a template, more than onedNTP/ddNTP can be used in the primer extension reaction.

In one embodiment, the invention provides a method, wherein only onedNTP, which corresponds to the nucleotide adjacent to the detectionprimer in the rare mutant nucleic acid molecule is used together withthe detection primer.

In another embodiment, the invention provides a method of detection ofnucleic acid molecules with a rare mutation comprising amplifying thenucleic acid sample with two primers that are designed toallele-specifically amplify the rare mutation containing nucleic acid,removing the excess dNTPs from the reaction after the amplificationreaction, performing the primer extension reaction with at least onedNTP or ddNTP, preferably dNTP, and a detection primer, which has beendesigned so that the 3′ end is immediately adjacent to the mutationsite, so that only the mutant nucleic acid will serve as a template tothe primer extension reaction when the corresponding dNTP or ddNTP isused, and detecting the primer extension reaction product, whereinpresence of the primer extension product after the primer extensionreaction indicates the presence of a nucleic acid with a rare mutation.

In one embodiment, two reactions are performed using two differentdetection primers, wherein the first detection primer is designed toamplify the sense strand so that the 3′ end of the primer annealsimmediately adjacent to the mutation site in the sense strand and in thesecond reaction the detection primer is designed to amplify theantisense strand so that the 3′ end of the primer anneals immediatelyadjacent to the mutation site in the antisense strand.

In yet another embodiment, the invention provides a method ofquantifying nucleic acid molecules with rare mutations comprising thesteps of amplifying a nucleic acid sample and a known amount of acontrol nucleic acid sample in the same reaction, wherein the controlnucleic acid sample has been designed to have the same sequence as therare mutation containing amplicon with the exception of at least one, 2,3, 4, 5-10, preferably only one nucleic acid difference immediatelyadjacent to the mutation site. The amplification is performed withprimers flanking the mutation site. After amplification, the excessdNTPs are removed and a primer extension reaction is performed using atleast one detection primer, which is designed so that the 3′ end of theprimer anneals immediately adjacent to the rare mutation site. Thedetection reaction is performed in the presence of dNTPs and/or ddNTPs.For example, at least one deoxynucleotide (dNTP), corresponding to themutant nucleoside immediately 3′ of the detection primer and twodideoxynucleotides (ddNTPs), which correspond to the nucleoside(s) thatdifferentiate the control from the rare mutant nucleic acid. The primerextension products are then detected, and because the amount of thecontrol originally added to the amplification reaction is known, theratio of the control and the rare mutant containing nucleic acidmolecules is used to determine the exact quantity of the mutant nucleicacid molecules in the sample. Preferably, only one dNTP is used.However, so long as the primer and dNTP/ddNTP combinations are designedso that the more common nucleic acid cannot be amplified in the primerextension reaction, the combination of dNTPs/ddNTPs may vary.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E show quantification of rare mutations. In the absence (FIG.1A), 20 fold excess (FIG. 1C) and 100 fold excess (FIG. 1D) of wildtypeDNA, the ratios of mutant DNA and the competitor DNA are very similar.In FIG. 1B, only 500 fold excess wildtype DNA was present and neithermutant nor competitor DNA was present. In FIG. 1E, 500 fold excesswildtype DNA, mutant DNA and competitor DNA were all present. Thesequences of the nucleic acid molecules shown in the FIG. 1 aredescribed in the Example.

FIG. 2 shows a schematic view of quantitative and allele-specificexpression analysis with real competitive PCR. A point mutation in thecDNA sequence is used as the marker for allele-specific gene expressionanalysis. The competitor is designed to have a synthetic mutation nextto the natural mutation and is used for quantitative gene expressionanalysis. Three extension products from the two cDNA sequences and thecompetitor have different molecular weights, and are detected byMALDI-TOF MS. The peak area ratios of these products representaccurately the concentration ratios of the two cDNAs and the competitor.Since the absolute quantity of the competitor is known, the absolutequantities of the two cDNA sequences can be readily calculated.

FIG. 3 shows a mass spectra for allele-specific expression analysis. (A)Interleukin 6 gene. Peaks are identified by C, T and S. C represents theallele where the polymorphic site has a C residue. T represents theallele where the polymorphic site has a T residue. S represents thecompetitor. The peak areas of C, T and S peaks are automaticallycomputed by the RT software package (SEQUENOM). The peak area ratiosrepresent the concentration ratios of the starting cDNA sequences andthe competitor. The peak frequencies are 0.209, 0.263 and 0.528 for peakC, T and S, respectively. (B) lexA gene. Peak S, G and C represent thecompetitor, the exogenous and the endogenous lexA gene, respectively.Without arabinose induction, only endogenous lexA gene expression wasseen. With modest arabinose induction, both the endogenous and exogenouslexA gene expression were seen. Without induction, the peak frequenciesare 0.601, 0.004 and 0.395 for peak S, G and C, respectively. Withinduction, the peak frequencies are 0.509, 0.075 and 0.416 for peak S, Gand C, respectively. (C) ABCD-1 gene. Mut and WT represent mutant andwildtype alleles, respectively. For Q672X, the peak frequencies are0.984 and 0.016 for peak Mut and WT, respectively. For S213C, the peakfrequencies are 0.187 and 0.813 for peak Mut and WT, respectively. ForS108W, the peak frequencies are 0.995 and 0.005 for peak WT and Mut,respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for detecting andquantifying rare mutations in a biological sample. The sample nucleicacid molecules that can be used in the methods of the present inventioninclude DNA, RNA and cDNA molecules. The present invention provides amethod for robustly detecting whether such rare mutations occur in abiological sample.

The term “mutation” as used throughout the specification is intended toencompass any and all types of functional and/or non-functional nucleicacid changes, including mutations and polymorphisms in the targetnucleic acid molecule when compared to a wildtype variant of the samenucleic acid region or allele or the more common nucleic acid moleculepresent on the sample. Such changes, include, but are not limited todeletions, insertions, translocations, inversions, and basesubstitutions of one or more nucleotides.

As used herein, polymorphism refers to a variation in the sequence of agene in the genome amongst a population, such as allelic variations andother variations that arise or are observed. Genetic polymorphismsrefers to the variant forms of gene sequences that can arise as a resultof nucleotide base pair differences, alternative mRNA splicing orpost-translational modifications, including, for example, glycosylation.Thus, a polymorphism refers to the occurrence of two or more geneticallydetermined alternative sequences or alleles in a population. Thesedifferences can occur in coding and non-coding portions of the genome,and can be manifested or detected as differences in nucleic acidsequences, gene expression, including, for example transcription,processing, translation, transport, protein processing, trafficking, DNAsynthesis, expressed proteins, other gene products or products ofbiochemical pathways or in post-translational modifications and anyother differences manifested among members of a population. A singlenucleotide polymorphism (SNP) refers to a polymorphism that arises asthe result of a single base change, such as an insertion, deletion orchange in a base.

A polymorphic marker or site is the locus at which divergence occurs.Such site may be as small as one base pair (an SNP). Polymorphic markersinclude, but are not limited to, restriction fragment lengthpolymorphisms, variable number of tandem repeats (VNTR's), hypervariableregions, minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats and other repeating patterns, simple sequencerepeats and insertional elements, such as Alu. Polymorphic forms alsoare manifested as different mendelian alleles for a gene. Polymorphismsmay be observed by differences in proteins, protein modifications, RNAexpression modification, DNA and RNA methylation, regulatory factorsthat alter gene expression and DNA replication, and any othermanifestation of alterations in genomic nucleic acid or organellenucleic acids.

The allelic form occurring most frequently in a selected population issometimes referred to as the wildtype form.

Diploid organisms may be homozygous or heterozygous for allelic forms. Adiallelic or biallelic polymorphism has two forms. A triallelicpolymorphism has three forms.

The term “rare mutation” as used herein and throughout the specificationis intended to describe a mutation in a nucleic acid molecule which ispresent in less than 40% of the nucleic acid molecules in the sample,preferably in less than 30%, 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, 1%,0.8%, 0.5%, 0.1%, 0.05%, 0.01, or less compared to one or more, morecommon nucleic acid variants, which are referred to throughout thespecification as the “wildtype” nucleic acid variants. In oneembodiment, the rare nucleic acid is present in the sample in amountless than 10%, preferably less than 1%. The sample may include one ormore rare mutations and there may also be one or more wildtype variantsin the nucleic acid sample.

The deoxynucleotides or dNTPs according to the present invention aredATP, dTTP, dCTP, or dGTP. The dideoxynucleotides or the terminatornucleotides (ddNTPs) are ddATP, ddTTP, ddCTP, or ddGTP. The dNTPs andddNTPs can also be labeled with, for example, different fluorescentdyes, or other labels, such as radioactive molecules, which do notinterfere with the DNA polymerase function in the primer extensionreaction. Differentially labeled dNTPs or ddNTPs can be used todifferentiate the alleles after the primer extension reaction. Suchlabels and the methods of preparing labeled dNTPs and ddNTPs are wellknow to one skilled in the art.

The terms “nucleic acid sample”, “nucleic acid molecule”, or “nucleicacid” as described throughout the specification are intended toencompass nucleic acids isolated from any biological material, e.g.,human, animal, plant, bacteria, fungi, protist, viruses, from tissuesincluding blood, hair follicles, or other tissues, such as skinbiopsies, cells or cell cultures, body excrements such as semen, saliva,stool, urine, amniotic fluid and so forth. The nucleic acids can also beisolated from foodstuff, drinks, clothes, soil and any other source,wherein detection of rare nucleic acids compared to a more common orwildtype variants of the same is needed. Nucleic acid molecules can beisolated from a particular biological sample using any of a number ofprocedures, which are well-known in the art, the particular isolationprocedure chosen being appropriate for the particular biological sample.

In one embodiment, the invention provides a method of detecting one ormore nucleic acids with a rare mutation comprising the steps ofamplifying a nucleic acid molecule with two primers flanking orsurrounding the mutation site, designing a detection primer so that the3′ end of the detection primer anneals immediately adjacent to a nucleicacid which is different in the mutant molecule compared to the morecommon wildtype variant of the same nucleic acid molecule, removing theexcess dNTPs after the amplification reaction, performing a primerextension reaction using the detection primer and at least one dNTP orddNTP, which corresponds to the nucleotide adjacent to the detectionprimer present in the rare mutant nucleic acid molecule and is notpresent in the background of the more common nucleic acid molecule(s) orvariant(s), wherein the presence of a primer extension product in thereaction indicates the presence of the nucleic acid with a raremutation. Preferably, only one dNTP or ddNTP is used in the primerextension reaction.

In one embodiment, the invention provides a method, wherein only onedNTP, which corresponds to the nucleoside adjacent to the detectionprimer in the rare mutant nucleic acid molecule is used together withthe detection primer.

For example, a nucleic acid molecule contains the following sequence:

[SEQ ID NO: 1]       5′TGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTaggggcagatagcagtga[A/T]GAGAGCGAGAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCACCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCC TG3′,

wherein [A/T] represents a base A to T mutation. T mutation occurs atlow frequencies, for example, less than about 30%, 20%, 15%, 10%, 8%,5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.1%, 0.05%, 0.01% or less. Therefore,in a biological sample, most nucleic acid molecules for the regiondepicted above have the A base and only a very small percentage of thenucleic acid molecules present in the biological sample have the T baseat the polymorphic site.

To detect the rare mutation, nucleic acids are isolated from the sourcematerial, such as tissues/cells/fluids or other sources of interest,using any of the widely adopted methods well known to one skilled in theart. Two PCR primers flanking the mutation site are shown as underlinedsequences in the above example, wherein only the sense strand is shown.The primers are designed to amplify the DNA region for both the wildtypeand mutant DNAs. To further increase the sensitivity of the method todetect rare known mutations, allele specific primers can be used topreferentially amplify the rare mutation containing nucleic acidmolecule(s). After the PCR, the excess nucleotides in the amplificationreaction are removed, for example, using a shrimp alkaline phosphataseor a spin column or any other method well known to one skilled in theart. A third primer, so called detection primer, which is shown in theabove example in small letters, is used in a base extension reaction.The third primer can also be designed from the opposite direction andthe two primers in two parallel reactions can be used to cross-validatethe results. It is important that the detection primer is designed sothat its 3′ end anneals immediately before a nucleoside which isdifferent in the rare mutant(s) compared to more common wildtype nucleicacid variant(s) because the methods of the present invention are basedon the premise of preferentially detecting the rare nucleic acidmolecules.

In the base extension reaction using the above presented example nucleicacid template, only ddTTP or alternatively, only dTTP is used so thatonly the mutant nucleic acid will be used as the template for the baseextension reaction. The detection of the oligonucleotide resulting fromthe primer extension reaction, i.e., aggggcagatagcagtga-ddT [SEQ ID NO.:2] indicates that the mutant allele is present.

Other combinations of ddNTP and dNTP can also be used as far as thewildtype nucleic acid(s) cannot be used as the template for the baseextension reaction.

The methods of the present invention can additionally be used to detectmore than one rare nucleic acid variant in the sample. For example, amultiplex PCR and a subsequent multiplex primer extension reaction canbe designed using the teachings of the present invention to detect atleast 2, 3, 4, 5, 6, 7, 8, 9, 10-15 or even more than 15 mutations inthe same reaction, as long as none of the wildtype, or more commonvariants of the respective nucleic acid targets can serve as a templatefor the detection primers in the primer extension reaction.

The methods of the present invention are useful, for example, indetecting a small population of nucleic acids with a known mutationamong a background of wildtype nucleic acid variants in, for example,early diagnosis or prognosis of cancer or malignant cell growth in anindividual. The methods of the present invention are also useful inproviding a means for early detection of malignant cells containing newor additional mutations which may be a result of treatment of themalignancies, such as appearance of multi drug resistance mutations inan individual with the proviso that these mutations are known or becomeknown through screening of new mutations before designing the detectionprimers.

The methods of the present invention also provide a useful tool todetect viral infections or emerging virus mutants in an individualinfected with a virus, such as human immune deficiency virus (HIV),during the treatment of the disease thereby allowing early adjustment intreatment as a response to occurrence of new virus mutations.

Due to their sensitivity, the methods of the present invention providean ideal tool to detect rare mutations in detection of the presence andquantification of the amount of the rare nucleic acid changes.Therefore, applications for the methods of the present inventioninclude, for example, early benign or malignant tumor detection,prenatal diagnostics particularly when using a blood sample from themother, early viral or bacterial disease detection or detection ofemerging strains of treatment resistant strains of bacteria or virusesin a target sample, environmental monitoring, monitoring of effects ofpharmaceutical interventions such as early detection of multi drugresistance mutations in cancer treatment. The methods of the presentinvention are also useful in detection of rare mutant nucleic acidpopulations in mosaic organisms or individuals or one of their tissuescomposed of cells of more than one genotype, for example, in diagnosisof mitochondrial diseases or inherited diseases, wherein the mutationoccurred after fertilization during early development of the embryo orfetus resulting in a mosaic genotype and consequently a mosaicphenotype.

The methods of the present invention are also useful in detection ofrare mutations in inherited diseases which result in reduction of thetranscript levels. It is sometimes easier to detect mutations from anRNA sample than from a genomic DNA sample. However, mutations causingsignificantly reduced transcript levels are often missed in thesescreens. The methods of the present invention can be used in detectingthe known transcript reducing mutations which can be considered “raremutations” because the mutant transcript population represents only asmall percentage of the nucleic acids in the target sample.

Detection of rare mutations using the methods of the present inventionalso provide tools for forensic nucleic acid sample analysis byproviding a system to reliably detect presence or absence of specificknown nucleic acid polymorphisms to provide evidence, for example, toinclude or exclude crime suspects.

Additionally, the methods of the present invention are useful indetection of rare extremely virulent or dangerous mutations inbiological agents, such as bacteria and viruses, that can be used as abiological warfare agents. As the knowledge of dangerous mutations inviruses and/or bacteria increases, the present invention providesmethods to detect small quantities of these abnormal mutants in a largerpopulation of wildtype or less virulent agents.

The present invention also provides that the method can be modified forgenotyping assays that might have an allele dropout problem. An alleledropout occurs when one allele is poorly amplified or detected, and aheterozygotic allele is mis-called as a homozygote. The dropout alleleis often, but not always, the allele that produces a higher molecularweight base extension product.

For example, if the allele with the T base at the SNP (single nucleotidepolymorphism) site is dropped out in a typical genotyping assay in theabove presented example nucleic acid, the method according to thepresent invention provides that the use of ddTTP only, or ddTTP and muchlower concentrations of other ddNTP/dNTP combinations, for the baseextension reaction, will result in preferential extension of the‘dropped-out’ allele, and therefore allele dropout is avoided.

The detection methods for detecting the primer extension products of thepresent invention can be any detection method which is capable ofdetecting the primer extension product after the primer extensionreaction. If the dNTP or ddNTP is labeled with a detectable marker orreporter such as a fluorescent or radioactively label or some otherdetectable chemical group, the detection method is based on detectingthe incorporation of the label into the primer extension product. Suchdetection methods include gel electrophoresis with laser detection orgel electrophoresis with detection of radioactivity, or other methodswell known to one skilled in the art.

A “reporter molecule”, as used herein, is a molecule which provides ananalytically identifiable signal allowing detection of a hybridizedprobe. Detection may be either qualitative or quantitative. Commonlyused reporter molecules include fluorophores, enzymes, biotin,chemiluminescent molecules, bioluminescent molecules, digoxigenin,avidin, streptavidin, or radioisotopes. Commonly used enzymes includehorseradish peroxidase, alkaline phosphatase, glucose oxidase andbeta-galactosidase, among others. Enzymes can be conjugated to avidin orstreptavidin for use with a biotinylated probe. Similarly, probes can beconjugated to avidin or streptavidin for use with a biotinylated enzyme.The substrates to be used with these enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase reporter molecules; forhorseradish peroxidase, 1,2-phenylenediamine, 5-aminosalicylic acid ortolidine are commonly used. Incorporation of a reporter molecule into aDNA probe can be by any method known to the skilled artisan, for exampleby nick translation, primer extension, random oligo priming, by 3′ or 5′end labeling or by other means (see, for example, Sambrook et al.Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y.1989).

Alternatively, the identified nucleic acids need not be labeled and canbe used to quantitate allelic frequency using a mass spectrometrytechnique described in Ding C. and Cantor C. R., 2003, Proc. Natl. Acad.Sci. U.S.A. 100, 3059-64, which is herein incorporated by reference inits entirety.

The preferred method for detecting the primer extension productscomprising the rare mutant nucleic acid is MALDI-TOF MS, using e.g.MASSARRAY™ system (Sequenom Inc., San Diego, Calif.).

In another embodiment, the invention provides a method of detectingnucleic acid molecules with a rare mutation comprising amplifying thenucleic acid sample with two primers that are designed toallele-specifically amplify the rare mutation containing nucleic acid,removing the excess dNTPs from the reaction after the amplificationreaction, performing the primer extension reaction with only one dNTP orddNTP, preferably dNPT, and a detection primer, which has been designedso that the 3′ end is immediately adjacent to the mutation site, so thatonly the mutant nucleic acid will serve as a template to the primerextension reaction when the corresponding dNTP or ddNTP is used, anddetecting the primer extension reaction product, wherein presence of theprimer extension product after the primer extension reaction indicatesthe presence of a nucleic acid with a rare mutation.

In one embodiment, two reactions are performed using two differentdetection primers, wherein the first detection primer is designed toamplify the sense strand so that the 3′ end of the primer annealsimmediately adjacent to the mutation site in the sense strand and in thesecond reaction the detection primer is designed to amplify theantisense strand so that the 3′ end of the primer anneals immediatelyadjacent to the mutation site in the antisense strand.

In yet another embodiment, the invention provides a method ofquantification of nucleic acid molecules with rare mutations comprisingthe steps of amplifying a nucleic acid sample and a known amount of acontrol nucleic acid sample in the same reaction, wherein the controlnucleic acid sample has been designed to have the same sequence as therare mutation containing amplicon with the exception of one nucleic aciddifference immediately adjacent to the mutation site. The amplificationis performed with primers flanking the mutation site. After theamplification, the excess of dNTPs are removed and a primer extensionreaction is performed using a detection primer, which is designed sothat the 3′ end of the primer anneals immediately adjacent to the raremutation site. The detection reaction is performed in the presence ofone deoxynucleotide (dNTP) and two dideoxynucleotides (ddNTPs): the dNTPcorresponds to the first nucleoside after the 3′ end of the detectionprimer in the nucleic acid with the rare mutation, the first ddNTPcorresponds to the nucleoside artificially created to the control whichdiffers from the nucleoside present in the rare mutant allele, and thesecond ddNTP corresponds to the nucleoside present in the rare mutantallele, preferably immediately after the mutation site. The primerextension products are then detected, and because the amount of thecontrol originally added to the amplification reaction is known, theratio of the control and the rare mutant containing nucleic acidmolecules is used to determine the exact quantity of the mutant nucleicacid molecules in the sample.

The standard nucleic acid can be prepared using any method of nucleicacid synthesis know to one skilled in the art, including, for example,chemical oligonucleotide synthesis, by cloning and targeted mutagenesis,or by PCR with mutagenized primers.

Oligonucleotide primers or standards may be synthesized using methodswell known in the art, including, for example, the phosphotriester (seeNarang, S. A., et al., 1979, Meth. Enzymol., 68:90; and U.S. Pat. No.4,356,270), phosphodiester (Brown, et al., 1979, Meth. Enzymol.,68:109), and phosphoramidite (Beaucage, 1993, Meth. Mol. Biol., 20:33)approaches. Each of these references is incorporated herein in itsentirety by reference.

In one embodiment, rolling circle amplification (RCA) is used. Rollingcircle amplification is an isothermal process for generating multiplecopies of a sequence. In rolling circle DNA replication in vivo, a DNApolymerase extends a primer on a circular template (Komberg, A. andBaker, T. A. DNA Replication, W. H. Freeman, New York, 1991). Theproduct consists of tandemly linked copies of the complementary sequenceof the template. RCA is a method that has been adapted for use in vitrofor DNA amplification (Fire, A. and Si-Qun Xu, Proc. Natl. Acad Sci.USA, 1995, 92:4641-4645; Lui, D., et al., J. Am. Chem. Soc.,1996,118:1587-1594; Lizardi, P. M., et al., Nature Genetics, 1998,19:225-232; U.S. Pat. No. 5,714,320 to Kool). RCA techniques are wellknown in the art, including linear RCA (LRCA). Any such RCA techniquecan be used in the present invention.

The methods of the present invention can be modified to utilize one ormore control or competitor nucleic acids to quantify the amount of oneor more rare mutant nucleic acid molecules in the same reaction.

The amount of the primer extension products is consequently measured byany of a variety of means, preferably by Mass Spectrometry (MALDI-TOF,or Matrix Assisted Laser Desorption Ionization-Time of Flight). InMALDI-TOF mass spectrometry, the peak area ratio between the productsfrom the standard and the nucleic acid of interest comprising the raremutation represents the ratio of the standard and the gene of interest.Since the concentration of the standard is known, the concentration ofthe nucleic acids with the rare mutation can be calculated.

Products of the primer extension reaction are detected and quantifiedusing methods including, but not limited to, MALDI-TOF massspectrometry, PYROSEQUENCING™, real time PCR, hybridization-basedtechniques, third wave invader assay, and fluorescence-based detectiontechniques.

In one preferred embodiment, the detection of the primer extensionproducts in the methods of the present invention is performed using theMALDI-TOF mass spectrometry, using, for example the MASSARRAY™ systemaccording to the manufacturer's instructions (Sequenom Inc., San Diego,Calif.).

Alternatively, an INVADER® assay can be used (Third Wave Technologies,Inc (Madison, Wis.)). This assay is generally based upon astructure-specific nuclease activity of a variety of enzymes, which areused to cleave a target-dependent cleavage structure, thereby indicatingthe presence of specific nucleic acid sequences or specific variationsthereof in a sample (see, e.g. U.S. Pat. No. 6,458,535). For example, anINVADER® operating system (OS), provides a method for detecting andquantifying DNA and RNA. The INVADER® OS is based on a “perfect match”enzyme-substrate reaction. The INVADER® OS uses proprietary CLEAVASE®enzymes (Third Wave Technologies, Inc (Madison, Wis.)), which recognizeand cut only the specific structure formed during the INVADER® process.The INVADER® OS relies on linear amplification of the signal generatedby the INVADER® process, rather than on exponential amplification of thetarget. This allows quantification of target concentration.

In the INVADER® process, two short DNA probes hybridize to the target toform a structure recognized by the CLEAVASE® enzyme. The enzyme thencuts one of the probes to release a short DNA “flap.” Each released flapbinds to a fluorescently-labeled probe and forms another cleavagestructure. When the CLEAVASE® enzyme cuts the labeled probe, the probeemits a detectable fluorescence signal.

In one embodiment, the primer extension products for the rare mutationsare detected using PYROSEQUENCING™ (Uppsala, Sweden), which isessentially sequencing by synthesis. A sequencing primer, designed toanneal directly next to the nucleic acid differing between the rare andthe common allele or the artificially produced quantification standardis first hybridized to a single stranded, PCR amplified, DNA templatecomprising both the target and the standard PCT product, and incubatedwith the enzymes, DNA polymerase, ATP sulfurylase, luciferase andapyrase, and the substrates, adenosine 5′ phosphosulfate (APS) andluciferin. One of four deoxynucleotide triphosphates (dNTP), forexample, corresponding to the nucleotide present in the standardtemplate, is then added to the reaction. DNA polymerase catalyzes theincorporation of the dNTP into the standard DNA strand. Eachincorporation event is accompanied by release of pyrophosphate (PPi) ina quantity equimolar to the amount of incorporated nucleotide.Consequently, ATP sulfurylase quantitatively converts PPi to ATP in thepresence of adenosine 5′ phosphosulfate. This ATP drives theluciferase-mediated conversion of luciferin to oxyluciferin thatgenerates visible light in amounts that are proportional to the amountof ATP. The light produced in the luciferase-catalyzed reaction isdetected by a charge coupled device (CCD) camera and seen as a peak in aPYROGRAM™. Each light signal is proportional to the number ofnucleotides incorporated and allows determination of the amount of thestandard nucleic acid sequence. Thereafter, apyrase, a nucleotidedegrading enzyme, continuously degrades unincorporated dNTPs and excessATP. When degradation is complete, another dNTP is added whichcorresponds to the dNTP present in the target template the amount ofwhich is to be determined. Finally, addition of dNTPs is performed oneat a time. Deoxyadenosine alfa-thio triphosphate (dATPαS) is used as asubstitute for the natural deoxyadenosine triphosphate (dATP) since itis efficiently used by the DNA polymerase, but not recognized by theluciferase. Because the amount of the standard added in the PCR isknown, the amount of the target can be calculated from the ratio of theincorporated dNTPs. For detailed information about reaction conditions,see, e.g. U.S. Pat. No. 6,210,891, which is herein incorporated byreference in its entirety.

The following illustrates quantification of concentration or copynumbers of rare alleles using the methods of the present invention. Thesequence is the same example as above:

[SEQ ID NO: 12]       5′TGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTaggggcagatagcagtga[A/T]{G/C}AGAGCGAGAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCACCTGACTCCTGAGGAGAAGTCTGCCGTTACT GCCCTG3′,

wherein all the notations are the same as above, except the {G/C}. TheG/C mutation is created to provide a detectable standard for thequantification reaction. In other words, a synthetic oligonucleotidewith the sequence as the following

GCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTAGGGGCAGATAGCAGTGA TCAGAGCGAGAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCACC[SEQ ID NO.: 3] is used as the internal standard for competitive PCR,wherein the bolded, underlined T represents the same nucleoside as inthe rare mutant nucleic acid and the C is created to provide adetectable difference between the rare mutant and the standard.

The competitor carries the T base as the rare mutation at the naturalpolymorphic site. In addition, it also has a C base, instead of the Gbase, at the position next to the polymorphic site.

PCR, excess dNTP removal using, for example, shrimp alkaline phosphatasetreatment, and consequently the base extension reaction are carried out.In the base extension reaction of the example, dTTP, ddGTP and ddCTPmixture is used. As a result, two extension products:aggggcagatagcagtgaTddG [SEQ ID NO.: 4] and aggggcagatagcagtgaTddC [SEQID NO.: 5] are produced. The first product comes from the rare mutationand the second product comes from the internal standard, the initialconcentration of which is known. The ratio of the two products can bequantified by, for example, MALDI-TOF mass spectrometry, or othertechniques, such as fluorescence measurement when ddCTP and ddGTP aretagged with different fluorescent groups.

EXAMPLE 1

Detection and quantification of rare mutation. Three DNA sequencesincluding wildtype (wt), mutant (mut) and a competitor or the standardwere used in this experiment. The sequences were:

WILDTYPE: [SEQ ID NO.: 6] 5′GTGGCAGATCTCTTCATGGTCTTCGGTGGCTTCACCACCAACCTCTACACCTCTCTCCATGGGTACTTCGTCTTTGG-3′ MUTANT: [SEQ ID NO.: 7]5′GTGGCAGATCTCTTCATGGTCTTCGGTGGCTTCACCACCATCCTCTACACCTCTCTCCATGGGTACTTCGTCTTTGG-3′ COMPETITOR: [SEQ ID NO.: 8]5′GTGGCAGATCTCTTCATGGTCTTCGGTGGCTTCACCACCATGCTCTACACCTCTCTCCATGGGTACTTCGTCTTTGG-3′

The competitor was used as an internal standard for mut DNAquantification. Wt DNA is used as the background DNA which exist at amuch higher concentration than mut DNA. The PCR primer sequences are:5′ACGTTGGATGTGGCAGATCTCTTCATGGTC-3′ [SEQ ID NO.: 9] and5′ACGTTGGATGCCAAAGACGAAGTACCCATG-3′ [SEQ ID NO.: 10]. The extensionprimer sequence was 5′CGGTGGCTTCACCACCA-3′ [SEQ ID NO.: 11]. Theextension ddNTP/dNTP mixture was dTTP/ddGTP/ddCTP.

Different mixtures of the three DNAs were co-amplified by PCR. ExcessdNTPs used in the PCR reaction were removed by shrimp alkalinephosphatase. Primer extension reaction was carried out using theextension primer and the extension ddNTP/dNTP mixture. FIGS. 1A-1Eillustrate the results of the MALDI-TOF mass spectrometric analysis ofthe primer extension products. In the absence of wildtype DNA (FIG. 1A),20 fold excess of wildtype DNA (FIG. 1C) and 100 fold excess of wildtypeDNA (FIG. 1D), the ratios of mutant DNA and the competitor DNA are verysimilar which well exemplifies that the method of the present inventionis capable of specifically amplifying the mutant allele and that therare mutation can be enriched to provide an efficient detection andquantification method for detecting rare mutations in the presence ofthe much more common background nucleic acid variant. In FIG. 1B, only500 fold excess wildtype DNA was present and neither mutant norcompetitor DNA was present. The figure illustrates the specificity ofthe system to amplify only the rare mutant and the added standard, orcompetitor nucleic acid. In FIG. 1E, 500 fold excess wildtype DNA,mutant DNA and competitor DNA were all present.

EXAMPLE 2

Matrix-assisted laser desorption ionization-time of flight massspectrometry (MALDI-TOF MS) was adapted for quantitative gene expressionanalysis [1]. This technique, dubbed as real competitive PCR, combinescompetitive PCR, primer extension reaction and MALDI-TOF MS. Afterisolation of RNA and reverse transcription, cDNA is spiked with asynthetic oligonucleotide (the competitor) with an identical sequenceexcept one single base roughly in the middle of the sequence to the cDNAof interest. The competitor and the cDNA of interest are co-amplified byPCR. Excess dNTPs are removed by shrimp alkaline phosphatase treatmentafter PCR. Then, a base extension reaction is carried out with anextension primer, a combination of three different ddNTPs and one dNTPand a ThermoSequenase. The base extension primer hybridizes right nextto the mutation site and either one or two bases are added for thecompetitor and the cDNA, yielding two oligonucleotide products withdifferent molecular weights (typically around 300 Da difference). In atypical molecular weight window of 4,000 to 9,000 Da, MALDI-TOF MS caneasily distinguish two oligonucleotides if they differ by more than 10Da. These two extension products are thus readily identified, and theratio of their concentrations is quantified by MALDI-TOF MS.

As shown in FIG. 2, when the synthetic mutation created in thecompetitor is close to a natural mutation site in the cDNA sequence,real competitive PCR can be used for accurate allele-specific geneexpression analysis. PCR is used to amplify the two cDNA sequences fromthe two alleles and the competitor. A base extension reaction with amixture of three different ddNTPs and one dNTP is used to generate three(instead of two in a typical real competitive PCR experiment)oligonucleotides for the two cDNAs and the competitor. The threeproducts are identified and their ratios are calculated based on theirpeak areas in the mass spectrum.

Since the amount of competitor spiked in is known, the absoluteconcentration of each of the two cDNAs can be easily calculated. Thus,it is possible to simultaneously quantify the gene expression levelsfrom the two alleles of one gene. The competitor and the two cDNAs arevirtually identical in sequence and are amplified with the samekinetics. The allele specificity is superior due to the high precisionof MALDI-TOF MS in molecular weight determination.

One example of allele-specific expression analysis by real competitivePCR is shown in FIG. 3A. A single nucleotide polymorphism (refSNP ID:rs2069849) located in exon 2 of the interleukin 6 gene is selected asthe marker for allele-specific expression. Complementary DNA (0.025 ng)prepared from the IMR-90 cell line (ATCC) was co-amplified with 5×10⁻²²Mol (301 copies) of the competitor. The oligonucleotide products fromthe base extension reaction were analyzed by MALDI-TOF MS. The peak arearatios represent accurately the concentration ratios of the two cDNAsand the competitor. Coefficient of variations (CV is defined as standarddeviation divided by the mean) for the relative frequencies of the threepeaks were 9.2%, 4.1% and 4.4% for four real competitive PCR replicates,indicating excellent precision. The interleukin 6 gene also shows modestskewing in allelic expression (98 copies of C allele was expressed, and136 copies of T allele was expressed, see FIG. 3A).

We next tested allele-specific expression of the lexA gene inEscherichia coli. Gene expression perturbation in E. coli was used forgene network studies [2]. Expression perturbation was achieved byintroducing an exogenous copy for each gene of interest in an inducibleexpression plasmid. The expression of each gene potentially in a generegulatory network was perturbed via the induction of the exogenous geneexpression, and the expression changes of other genes were analyzed.These perturbed gene expression levels were then fed into a multiplelinear regression algorithm to estimate the network interactions. Thisapproach is a powerful tool for functional genomics analysis. However,self-regulatory interactions such as positive and negativeself-feedbacks can only be resolved by measuring the exogenous andendogenous gene expression separately. In the original study on the E.coli network, a reporter gene (luciferase), expressed under identicalconditions as the gene of interest, was used to estimate the exogenousgene expression. However, this estimate is likely to be inaccurate sincethe expression level of the luciferase gene is likely to be differentfrom the exogenous genes, even when they are under the control of thesame promoter. A method to directly and separately quantify theexpressions of the exogenous and the endogenous gene is needed to obtainsignificantly more accurate estimates of self-regulatory interactions ingene networks.

To this end, an exogenous lexA was introduced into E. coli via thepBADX53 vector. The exogenous lexA gene is distinguishable from theendogenous lexA gene by a silent mutation (TCC to TCG silent mutation atcodon 103). The exogenous lexA expression was induced with arabinose.Without arabinose, only endogenous lexA transcript was detected (FIG.3B). With an intermediate arabinose induction, exogenous lexA wasexpressed at about 20% level compared with the endogenous lexA (FIG.3B).

EXAMPLE 3

In the third example, we tested allele-specific expression of the ABCD-1gene (located on the X chromosome) involved in X-linkedadrenoleukodystrophy (XALD). The manifestation of symptoms in XALDcarriers was previously shown to be associated with a higher degree ofnon-random X chromosome inactivation [3]. A non-random X chromosomeinactivation is likely to cause a preferential expressiondown-regulation of one of the ABCD-1 allele. If the wildtype allele isinactivated, the mutant allele will be predominantly expressed. Thus,the individual might show symptoms similar to a homozygous mutant. Xchromosome inactivation studies can only provide a genome-wide, indirectpicture while direct allele-specific gene expression can provide thedirect link between gene expression and disease manifestation. We thuscarried out allele-specific gene expression for three carriers withthree different ABCD-1 mutations (S108W, S213C and Q672X). The S108Wcarrier showed predominant (>99%) mutant allele expression while theS213C and Q672X showed predominant wildtype allele (89% and >99%,respectively) expression (FIG. 3C). This result is in completeconcordance with results obtained previously [3].

These examples demonstrate quantitative and allele-specific geneexpression analysis with real competitive PCR. The allele specificityfor gene expression analysis used is the superior molecular weightdetermination ability of the MALDI-TOF MS technology. Highly precise (CV4%-9%) and absolute gene expression analysis is achieved. In addition,the real competitive PCR used the highly automated MassARRAY system(SEQUENOM), and is ideal for high-throughput (7000reactions/day/instrument) analysis. The high-throughput and low costfeatures of this technique can easily be exploited in large-scaleallele-specific expression studies.

MATERIALS AND METHODS

cDNA and Oligonucleotides

Interleukin 6 Gene Expression Analysis

Complementary DNA for interleukin 6 gene expression analysis wasprepared from cell line IMR-90 (ATCC). The PCR primer sequences for theinterleukin 6 gene expression analysis are:

[SEQ ID NO: 13] 5′-ACGTTGGATGGCAGGACATGACAACTCATC-3′ and [SEQ ID NO: 14]5′-ACGTTGGATGCCATGCTACATTTGCCGAAG-3′.The extension primer sequence is 5′-CGCAGCTTTAAGGAGTT-3′ [SEQ ID NO:15]. The synthetic competitor sequence is5′-GCCCATGCTACATTTGCCGAAGAGCCCTCAGGCTGGACTGCATAAACTCCTTAAAGCTGCGCAGAATGAGATGAGTTGTCATGTCCTGCAG-3′[SEQ ID NO: 16]. All oligonucleotides were purchased from Integrated DNATechnologies (Coralville, Iowa). The synthetic competitor was PAGEpurified by the vendor and absorbance at 260 nm was measured in ourlaboratory.

lexA Gene Expression Analysis

RNA samples for lexA gene expression analysis were provided by Dr.Timothy Gardner (Boston University). The exogenous lexA gene has a TCCto TCG silent mutation at codon 103 so that it can be distinguished fromthe endogenous lexA gene. The exogenous lexA gene was cloned in thevector pBADX53. Bacterial culture and RNA extraction were carried out aspreviously described [10]. The PCR primer sequences for the lexA geneexpression analysis are, 5′-ACGTTGGATGGCGCAACAGCATATTGAAGG-3′ [SEQ IDNO: 17] and 5′-ACGTTGGATGACATCCCGCTGACGCGCAGC-3′ [SEQ ID NO: 18]. Theextension primer sequence is 5′-ATCAGCATTCGGCTTGAATA-3′ [SEQ ID NO: 19].The synthetic competitor sequence is

[SEQ ID NO: 20] 5′- ACATCCCGCTGACGCGCAGCAGGAAATCAGCATTCGGCTTGAATATGGAAGGATCGACCTGATAATGACCTTCAATATGCTGTTGCGC-3′.

The synthetic competitor was PAGE purified by the vendor and absorbanceat 260 nm was measured in our laboratory.

ABCD-1 Gene Expression Analysis

Complementary DNA and genomic DNA samples for ABCD-1 gene expressionanalysis were prepared as previously described [11]. Three ABCD-1carriers, S108W, S213C and Q672X, were used in this study. PCR primersfor the three mutations are: 5′-ACGTTGGATGAGCAGCTGCCAGCCAAAAGC-3′ [SEQID NO: 21] and 5′-ACGTTGGATGACTCGGCCGCCTTGGTGAG-3′ [SEQ ID NO: 22] forS108W, 5′-ACGTTGGATGTAGGAAGTCACAGCCACGTC-3′ [SEQ ID NO: 23] and5′-ACGTTGGATGAACCCTGACCAGTCTCTGAC-3′ [SEQ ID NO: 24] for S213C, and5′-ACGTTGGATGTCCCTGTGGAAATACCACAC-3′ [SEQ ID NO: 25] and5′-ACGTTGGATGAGTCCAGCTTCTCGAACTTC-3′ [SEQ ID NO: 26] for Q672X. Theextension primers are: 5′-GGCGGGCCACATACACC-3′ [SEQ ID NO: 27] forS108W, 5′-AGTGGCTTGGTCAGGTTG-3′ [SEQ ID NO: 28] for S213C and5′-AATACCACACACACTTGCTA-3′ [SEQ ID NO: 29] for Q672X.

Real Competitive PCR

Real competitive PCR was carried out as was previously described [9].

Step 1: PCR Amplification

Each PCR reaction contains 1 μL diluted cDNA (0.025 ng/μL), 0.5 μL 10×HotStar Taq PCR buffer, 0.2 μL MgCl₂ (25 mM), 0.04 μL dNTP mix (25 mMeach), 0.02 μL HotStar Taq Polymerase (50 U/μL, Qiagen), 0.1 μLcompetitor oligonucleotide (5×10⁻⁹ μM), 1 μL forward and reverse primer(1 μM each) and 2.14 μL ddH₂O. The PCR condition was: 95° C. for 15 minfor hot start, followed by denaturing at 94° C. for 20 sec, annealing at56° C. for 30 sec and extension at 72° C. for 1 min for 45 cycles, andfinally incubated at 72° C. for 3 min.

Step 2: Shrimp Alkaline Phosphatase Treatment

PCR products were treated with shrimp alkaline phosphatase to removeexcess dNTPs. A mixture of 0.17 μL hME buffer (SEQUENOM), 0.3 μL shrimpalkaline phosphatase (SEQUENOM) and 1.53 μL ddH₂O was added to each PCRreaction. The reaction solutions (now 7 μL each) were incubated at 37°C. for 20 min, followed by 85° C. for 5 min to inactive the enzyme.

Step 3: Single Base Extension Reaction

For each base extension reaction, 0.2 μL of selected ddNTPs/dNTP mixture(SEQUENOM), 0.108 μL of selected extension primer, 0.018 μL ofThermoSequenase (32 U/μL, SEQUENOM) and 1.674 μL ddH₂ 0 were added. Thebase extension condition was, 94° C. for 2 min, followed by 94° C. for 5sec, 52° C. for 5 sec and 72° C. for 5 sec for 40 cycles. TheddNTPs/dNTP mixtures are: ddATP/ddCTP/ddGTP/dTTP for interleukin 6 andABCD-1 Q672X, ddTTP/ddCTP/ddGTP/dATP for lexA, andddATP/ddCTP/ddTTP/dGTP for ABCD-1 S108W and S213C.

Step 4: Liquid Dispensing and MALDI-TOF MS

The final base extension products were treated with SpectroCLEAN(SEQUENOM) resin to remove salts in the reaction buffer. This step wascarried out with a Multimek (Beckman) 96 channel auto-pipette and 16 μLresin/water solution was added into each base extension reaction, makingthe total volume 25 μL. After a quick centrifugation (2,500 rpm, 3 min)in a Sorvall legend RT centrifuge, approximately 10 nL of reactionsolution was dispensed onto a 384 format SpectroCHIP (SEQUENOM)pre-spotted with a matrix of 3-hydroxypicolinic acid (3-HPA) by using aMassARRAY Nanodispenser (SEQUENOM). A modified Bruker Biflex MALDI-TOFmass spectrometer was used for data acquisitions from the SpectroCHIP.Mass spectrometric data were automatically imported into theSpectroTYPER (SEQUENOM) database for automatic analysis such as noisenormalization and peak area analysis.

REFERENCES

-   -   1. Ding C, Cantor C R. A high-throughput gene expression        analysis technique using competitive PCR and matrix-assisted        laser desorption ionization time-of-flight MS. Proc Natl Acad        Sci USA 2003; 100:3059-3064.    -   2. Gardner T S, di Bernardo D, Lorenz D, Collins J J. Inferring        genetic networks and identifying compound mode of action via        expression profiling. Science 2003; 301:102-105.    -   3. Maier E M, Kammerer S, Muntau A C, Wichers M, Braun A,        Roscher A A. Symptoms in carriers of adrenoleukodystrophy relate        to skewed X inactivation. Ann Neurol 2002; 52:683-688.

The above-cited references and those reference cited throughout thespecification are herein incorporated by reference in their entirety.

1. A method of detecting nucleic acids with a rare mutation, whereinsaid rare mutation includes any change from the wildtype sequenceincluding polymorphisms, comprising the steps of; a) amplifying anucleic acid molecule with primers flanking the rare mutation site(s);b) removing the excess dNTPs after the amplification reaction; c)performing a primer extension reaction using a detection primer(s) whichis designed so that the 3′ end of the detection primer is immediatelyadjacent to a nucleic acid which differentiates the wildtype from themutant nucleic acid molecule, and at least one dNTP or ddNTP, whichcorresponds to a nucleoside adjacent to the detection primer in the raremutant nucleic acid molecule; and d) detecting the presence of theprimer extension product(s) after the primer extension reaction and/orthe consumption of dNTP, wherein the presence of a primer extensionproduct in the reaction or the consumption of dNTP indicates thepresence of the nucleic acid with a rare mutation.
 2. The method ofclaim 1, wherein the consumption of dNTP is detected usingpyrosequencing.
 3. The method of claim 1, wherein only one dNTP or ddNTPcorresponding to a nucleoside differentiating the rare nucleic acidvariant from the more common nucleotide variant(s) is used.
 4. Themethod of claim 3, wherein only one dNTP is used.
 5. The method of claim1, wherein a mixture of dNTP(s)/ddNTP(s)ddNTP(s) are used, wherein noneof the dNTPs or ddNTPs can also be used for the extension of thewildtype DNA.
 6. The method of claim 1 wherein the step of detecting thepresence of the primer extension product further includes measuring theamount of the primer extension product in the reaction.
 7. A method ofdetecting nucleic acid molecules with a rare mutation comprising thesteps of: a) amplifying the nucleic acid sample with primers that aredesigned to allele-specifically amplify the rare mutation containingnucleic acid, wherein the rare mutation represents any change from thewildtype nucleic acid including polymorphisms; b) removing the excessdNTPs from the reaction after the amplification reaction; c) performingthe primer extension reaction with at least one dNTP or ddNTP,preferably dNTP, and a detection primer(s), which has been designed sothat the 3′ end anneals immediately adjacent to the mutation site, sothat only the mutant nucleic acid will serve as a template to the primerextension reaction when the corresponding dNTP(s) or ddNTP(s) are used;and d) detecting the primer extension reaction product(s), whereinpresence of the primer extension product(s) after the primer extensionreaction indicates the presence of a nucleic acid with a rare mutation.8. The method of claim 1, wherein parallel primer extension reactionsare performed using two different detection primers, wherein the firstdetection primer is designed to amplify the sense strand so that the 3′end of the primer anneals immediately adjacent to the mutation site inthe sense strand and in the second reaction the detection primer isdesigned to amplify the antisense strand so that the 3′ end of theprimer anneals immediately adjacent to the mutation site in theantisense strand.
 9. A method of determining the concentration or thecopy number of nucleic acid molecules with rare mutations comprising thesteps of: a) amplifying a nucleic acid sample and a known amount of acontrol competitive nucleic acid standard sample in the same reaction,wherein the control nucleic acid sample has been designed to have thesame sequence as the rare mutation containing amplicon with theexception of one nucleic acid difference immediately adjacent to themutation site, with primers flanking the mutation site; b) removing theexcess dNTPs; c) performing a primer extension reaction using adetection primer(s), which is designed so that the 3′ end of the primeranneals immediately adjacent to the rare mutation site and in thepresence of at least one deoxynucleotide (dNTP) and twodideoxynucleotides (ddNTPs), wherein the dNTP corresponds to the firstnucleoside after the 3′ end of the detection primer in the nucleic acidwith the rare mutation, the first ddNTP corresponds to the nucleosideartificially created to the control which differs from the nucleosidepresent in the rare mutant allele, and the second ddNTP corresponds tothe nucleoside present in the rare mutant allele immediately after themutation site; d) detecting the production of primer extension productsand/or consumption of ddNTP; and e) determining the ratio of theamplified rare mutant, wherein the rare mutant includes any change fromthe wildtype including polymorphisms and the standard competitor andcalculating the concentration or copy number of the rare mutant nucleicacid variant in the original sample base on the known amount of thecompetitor initially added to the amplification reaction in the step a).10. The method of claim 9, wherein a mixture of dNTP(s)/ddNTP(s) areused, wherein none of the dNTPs or ddNTPs can also be used for theextension of the wildtype DNA, and the extension product from the raremutant and the control DNA can be distinguished.
 11. The method of claim9, wherein the consumption of ddNTPs is quantified. 12 The method ofclaim 7, wherein parallel primer extension reactions are performed usingtwo different detection primers, wherein the first detection primer isdesigned to amplify the sense strand so that the 3′ end of the primeranneals immediately adjacent to the mutation site in the sense strandand the second reaction the dection primer is designed to amplify theantisense strand so that the 3′ end of the primer anneals immediatelyadjacent to the mutation site in the antisense strand.